In humans, four important glycoprotein hormone heterodimers (LH, FSH, TSH and CG) have differing β subunits but identical α subunits. Three of these hormones are directly related to fertility, and the fourth, TSH, is indirectly so related. Three of these hormones, LH, FSH and TSH, are present in virtually all other vertebrate species; CG has so far been found only in primates and in the placenta and urine of pregnant mares.
PCT application WO 90/09800, published 7 Sep. 1990 and incorporated herein by reference, describes a number of modified forms of these hormones. One important modification is a C-terminal extension of the β subunit by the carboxy terminal peptide (CTP) of human chorionic gonadotropin or a variant thereof. CTP is the sequence of amino acids extending from any one of positions 112–118 to position 145 of the β subunit of human chorionic gonadotropin. PCT publication WO 94/24148 published 27 Oct. 1994 and incorporated herein by reference describes modifying these hormones by addition of the CTP at locations other than the C-terminus as well as variants of the CTP itself which are shorter than the sequence defined above.
PCT publication WO 91/16922 published 14 Nov. 1991 describes a number of chimeric and otherwise modified forms of the heterodimeric glycoprotein hormones. This disclosure is incorporated herein by reference as well. In addition, two PCT publications describe single chain forms of these hormones wherein the α and β subunits are covalently linked to result in a fusion peptide of the formula β(linker)nα, or α(linker)nβ, wherein n is 0 or 1 and α and β represent the respective subunits of these hormones. These publications, WO 95/22340 published 24 Aug. 1995 and WO 96/05224 published 22 Feb. 1996 are incorporated herein by reference.
Forms of the single chain glycoprotein hormones in which the number of cystine bridges has been depleted are disclosed in U.S. Pat. No. 6,693,074 filed 19 Sep. 1997 and incorporated herein by reference.
PCT publication WO 99/25849 published 27 May 1999 and incorporated herein by reference describes covalent single chain forms of the glycoprotein hormones which contain two β subunits. The two β subunits may be the same or different and may be coupled through linkers to each other and to a common α subunit. These compounds are of the formulas β1-(linker1)m-α-(linker2)n-β2;β1-(linker1)m-β2-(linker2)n-α; andα-(linker1)m-β1-(linker2)n-β2.
the two β subunits are different, the single chain forms are multifunctional.
In addition, PCT publication WO 00/23473 published 27 Apr. 2000 and incorporated herein by reference also describes modified forms of these single chain hormones wherein one of the β subunits is coupled covalently, optionally through a linker to the α subunit while a second β subunit is non-covalently associated with the single chain form.
U.S. Pat. No. 6,689,365 filed 5 May 2000 and incorporated herein by reference describes a subgenus of single chain forms of the formulaFSHβ-(linker1)n1-LHβ(1−x)-(linker2)n2-α                wherein LHβ(1−x) refers to the LHβ subunit optionally containing deletions of up to 7 amino acids from the carboxy terminus and each linker is an amino acid sequence which is flexible and hydrophilic; each n is 0 or 1. By adjusting the lengths of the linkers, especially that between the LHβ subunit and α, the ratio of FSH to LH activity can be fine-tuned.        
Thus, while the art describes single chain multifunctional glycoprotein hormone forms, the multifunctionality has been limited to that ascribable to only two of the glycoprotein hormone heterodimers. It has now been found that single chain forms can be constructed wherein the activity of at least three of the hormones can be exhibited based on a single molecule. The relative degrees of these activities can be adjusted by controlling the spacing within the construct.